Somatic gene therapy of bone marrow stem cells has been proposed as an alternative to conventional therapies of AIDS. Successful inhibition of HIV replication using ribozymes, antisense oligonucleotides, RNA-decoy strategies and dominant-negative mutants has been reported. We have developed a new technology for the efficient generation of genetic suppressors represented by antisense RNA and dominant-negative mutants. It is based on the functional selection of genetic suppressor elements (GSEs) from a random fragment library generated from a gene or a genome of interest. This approach has been validated in two model systems: The selection of GSEs, derived from the lambda phage genome, that conferred bacterial resistance to phage-mediated lysis and-also the isolation of USEs from the human topoisomerase II gene which conferred etoposide resistance to human cells. We have generated USE libraries from the HIV-l genome and have selected putative USEs capable of protecting human cells from HIV-mediated cytopathicity. The aim of this proposal is to fully characterize the current pool of anti-HIV USE candidates by further selection and to also develop an alternative GSE selection procedure that will address limitations identified in preliminary studies. In addition, we will evaluate "GSE-cassettes" containing multiple, characterized, anti-HIV elements that inhibit different stages of the viral life cycle. This proposal will identify potential candidates for somatic gene therapy of HIV infected cells. PROPOSED COMMERCIAL APPLICATION: We have developed a new technology for the efficient generation of genetic suppressors represented by antisense RNA and dominant-negative mutants. It is based on the functional selection of genetic suppressor elements (USEs) from a random fragment library generated from a gene or a genome of interest. The aim of this proposal is to fully characterize the current pool of anti-HIV USE candidates by further selection and to also develop an alternative USE selection procedure that will address limitations identified in preliminary studies.